Location: Wheat, Sorghum and Forage Research
2023 Annual Report
Objectives
Objective 1: Identify and manipulate the genetic and biochemical mechanisms controlling lignin deposition to develop improved sorghum germplasm for bioenergy and forage uses.
Subobjective 1A: Characterize the effects six recently identified brown midrib mutants (bmr) on phenylpropanoid metabolism and lignin deposition.
Subobjective 1B: Evaluate ways to increase lignin deposition and alter phenolic composition of biomass through overexpression of monolignol genes.
Objective 2: Identify and manipulate the genetic and biochemical mechanisms controlling starch and phosphorus composition of grain to develop novel sorghum traits for food, biofuel and livestock production.
Subobjective 2A: Identify and characterize mutants that alter phosphorus composition and reduce phytate in grain.
Subobjective 2B: Develop germplasm with altered starch composition and content in grain.
Objective 3: Identify resistance to fungal pathogens in lignin modified sorghum germplasm for development of stalk rot-resistant grain, bioenergy, and forage sorghums.
Subobjective 3A: Determine the responses of sorghum lines with six recently identified bmr mutants to stalk pathogens.
Subobjective 3B: Assess impact of the stalk pathogen Fusarium thapsinum on sorghum with altered the monolignol synthesis.
Subobjective 3C: Determine the response of sorghum stalk moisture phenotypes on stalk rot pathogens.
Subobjective 3D: Determine whether beneficial microorganisms increase protection of bmr mutants against root and stalk pathogens.
Objective 4: Identify resistance interactions between sorghum grain with novel composition and fungal pathogens for food, fuel, and feed uses.
Subobjective 4A: Determine whether pericarp pigments provide protection against grain pathogens.
Subobjective 4B: Determine whether grain tannins prevent fungal infection.
Approach
Sorghum (Sorghum bicolor) is a climate resilient crop, which is capable of providing grain and forage (biomass) to both the existing agricultural markets and the emerging bioenergy markets in the United States. To compete in these markets, compositional improvements to both sorghum grain and forage are needed and an understanding how these changes affect the fungal pathogens of sorghum. The objectives of this project will focus on the genetic, biochemical, and physiological mechanisms affecting the composition of sorghum biomass and grain. Efforts will result in sorghum with altered lignin content and/or composition of biomass, and increased starch content and reduced phytate content of grain for improved bioenergy conversion, livestock utilization and human nutrition. The impacts of fungal pathogens on sorghum with compositionally modified biomass and grain will be determined. Sorghum germplasm with desirable traits enhancing sorghum biomass and grain utilization will be developed, fully characterized, released and deposited into USDA–ARS National Plant Germplasm System (NPGS) for use by public and private sector plant breeders for developing improved hybrids and cultivars. The project consists of three integrated components: germplasm development, molecular biology, and plant pathology. Molecular and conventional methodologies will be utilized, and the project scale will range from gene-level to field-level. The project also has extensive formal and informal collaborations enhancing our ability to conduct this research. Anticipated products include improved sorghum germplasm for the sorghum seed industry with value-added traits and biotic stress tolerance, and tools to assess these biological pathways and fungal pathogen responses of sorghum.
Progress Report
This five-year project ended in February of 2023, and research will continue under the new project number 3042-21220-033-000D, Genetic Improvement of Sorghum for Biomass, Grain, and Disease Resistance. The following key accomplishments were achieved over the past 5 years.
Lignin is a key component of plant cell walls that reduces the efficiency of bioenergy conversion and forage digestibility in livestock. In Objective 1, a transcription factor, SbMyb60 was shown to direct sorghum metabolism toward lignin deposition, which included amino acid phenylalanine and the required cofactors leading toward lignin synthesis. SbMyb60 affects biochemical pathways that also lead to lignin synthesis. The overexpression of ferulate 5-hydroxylase (F5H) and caffeoyl-CoA 3-O-methyltransferase (CCoAOMT) genes changed the cell wall composition without affecting plant yield. F5H overexpression alter the lignin composition of sorghum biomass, while CCoAOMT increased phenolic compounds within cell wall without affecting lignin. This research demonstrated new ways to change the cell wall composition of sorghum biomass, which may lead to the production of renewable chemicals from specific lignin component or related compounds.
The brown midrib (bmr) mutants have long been associated with plants impaired in their ability to synthesize lignin. In sorghum, the previously characterized brown midrib loci were all shown to encode enzymes in the monolignol pathway. In Objective 1, sorghum Brown midrib 30 (Bmr30) gene was discovered to encode a flavonoid enzyme required for lignin deposition, chalcone isomerase. This discovery linked plant purple pigments and lignin through a compound called tricin, which was recently been shown to be a component of lignin in grasses like sorghum. The Bmr19 gene was shown to encode an enzyme critical for the synthesis of the cofactor s-adenosylmethionine (SAM), which is required cofactor for monolignol synthesis. The bmr12 plants were discovered to have altered roots as compared to normal sorghum. Further analyses showed that bmr12 plants were primed to respond to drought even under well-watered conditions, which increase drought tolerance. Overall, these studies demonstrated new ways to reduce lignin and improve forage, bioenergy and green chemistry utilization in the climate resilient feedstock sorghum.
Sorghum grain is an important food and feed source throughout the world. However, phytate is an antinutritional phosphorus compound found in the grain. In Objective 2, sorghum mutants were identified with reduce levels of phytate. Grain from normal and waxy sorghum, a mutant affecting starch composition, was examined for its effects on the human colon microbiome using in vitro experiments. The normal sorghum starch is more resistant to digestion, which led to increased levels of bacteria beneficial to human health compared to waxy grain. Together these studies identified both viable and non-viable option to improve the nutritional value of sorghum grain for both humans and animals.
Stalk pathogens can be particularly devastating to sorghum production, and identifying sources of resistance is challenging because numerous types of fungi are responsible for stalk rot diseases. It is important to assess newly developed germplasm for resistance to stalk pathogens before deployment. A potential control measure to prevent stalk rot pathogens from infecting sorghum is beneficial to soil microorganisms. In Objective 3, potentially beneficial microorganisms were applied to sorghum seeds, and then germination and seedling size were measured in two types of sterilized field soil. The seeds tested included bmr6, bmr12,the double mutant, and their normal counterparts, which were treated with three fluorescent Pseudomonas bacterial strains, two of which showed promising results in previous tests of biological control agents for stalk rot. One strain also increased seedling growth under drought conditions. A set of 16 bacterial strains that associate with sorghum roots and soil, and inhibit pathogen growth, were characterized by sequencing with next-generation technology. We determined that four of these strains had a suite of genes that could indicate superior biological control capabilities such as competitiveness with other microorganisms, plant root colonization, drought survival, and plant growth promotion. The cell wall polymer lignin has been implicated as a defense against pathogens including stalk rots, and altering its content and composition may improve sorghum for forage and bioenergy uses. In Objective 3 we tested six novel bmr mutants decreased in lignin content, incorporated into three elite commonly-used sorghum backgrounds for susceptibility to Fusarium stalk rot and charcoal rot pathogens, and determined that these lines were not more susceptible to the tested pathogens than normal lines. Two sets of mutant lines were also tested for stalk diseases under drought conditions, which did not alter their susceptible to the pathogens. In one of the three backgrounds, the normal line was highly susceptible to charcoal rot under drought conditions, but the two mutant lines were resistant. In Objective 3 we also tested lines with bmr6, bmr12 and bmr2 and their corresponding overexpression lines for responses to the two stalk diseases under adequate water and drought conditions. These were as resistant as the normal lines to the two stalk pathogens. These results demonstrate that altering lignin synthesis does not increase susceptibility to these potentially devastating pathogens of sorghum including under drought conditions, which is important information for development of forage and bioenergy sorghums. The sorghum D locus controls whether sorghum stalks are dry or juicy, which can improve post-harvest drying of forage, but may affect interactions with stalk pathogens, although no such studies have appeared in the scientific literature. For Objective 3, we examined this potential interaction using a series of lines with either functional or non-functional alleles at D and Bmr6 loci for response to Fusarium stalk rot and charcoal rot pathogens. In greenhouse assessments, there were no significant differences in the diseases tested, which indicated that stalk composition controlled at the D locus does not affect sorghum stalk disease responses. In field assessments of Fusarium stalk rot under adequate water, the juicy line was more susceptible than the dry (normal) line, but the juicy bmr6 line was as resistant as the dry bmr6 line. There was no differences between the dry and juicy lines under dryland conditions however. Together these studies indicate that in combination with lignin modified lines, manipulation of stalk moisture content for forage or bioenergy uses may not alter susceptibility to stalk pathogens in some environments, but the bmr6 in combination with the juicy stalks may increase resistant compared their dry counterpart in all environments.
Fungal pathogens also infect sorghum grain, which may render it unusable as food or feed due to the presence of fungal toxins. The phenolic pigments of sorghum grain affect its end-uses, but they are also sources of antioxidants and may help defend against grain molds. In Objective 4, sorghum grain was grown in both Texas (high disease pressure) and Nebraska (moderate disease pressure) to assess the role of pigments in grain mold resistance. Fungi have been isolated and identified using morphological characteristics; molecular identification is on-going to identify strains with the potential to be pathogenic and toxigenic. Understanding the role of grain pigments is critical for controlling grain mold, because people consume both unpigmented and pigmented sorghum grain. Tannins (polyphenols) are also deposited in the outer layer of some sorghum grain. Tannins may also reduce mold infection, lines with or without tannins in a tall and a short background were grown at two locations in Nebraska over two years to determine whether tannins affect fungal infection. The results indicated that tannins can protect the grain from pathogenic and toxigenic fungi under dryland conditions, but not under irrigation. Because sorghum is often grown under marginal conditions, this result suggests that tannin plants would be superior in protecting grain against contamination with pathogenic or toxigenic fungi.
Accomplishments
1. Stalk rot-resistant sweet sorghum line responds to wounding with defense compounds and effective defense signaling. Charcoal rot and Fusarium stalk rot threaten sweet sorghum production, which is used to make molasses syrup, ethanol, and other products, but these diseases can reduce yield and diminish syrup quality. These pathogens enter the plant through wounds and cause stalk destruction resulting in flattened stands of plants. The best way to control these diseases is to identify sweet sorghum lines that are resistant to both diseases. ARS researchers in Lincoln, Nebraska, showed that the sweet sorghum variety M81-E was more resistant to both diseases than the sweet sorghum variety Colman. Using state-of-the-art molecular techniques, researchers identified the plant compounds and the genes induced in response to the charcoal rot infection, which showed M81-E activated sorghum defense pathways more rapidly than Colman did. These genes and pathways identified in this study are potential targets for developing new disease-resistant sweet sorghums. Discovering new mechanism to increase stalk rot resistance will increase harvestable yield and benefit both sweet sorghum producers
2. Recently identified Sorghum brown midrib lines were not susceptible to stalk rot pathogens under water deficit. In the U.S., the stalk diseases Fusarium stalk rot and charcoal rot cause significant losses of both sorghum biomass and grain due to flattened stands of plants, and are especially pervasive during flowering under limited water conditions. ARS scientists at Lincoln, Nebraska, developed brown midrib (bmr) plants in three different sorghum varieties from newly characterized alleles, which are impaired in lignin synthesis. Two lines, bmr29 and bmr31 lines were tested using a method developed to simulate drought in the greenhouse. The normal variety was highly susceptible to charcoal rot under drought conditions, but the bmr29-1 and bmr31-1 lines were as resistant to these conditions as the adequately watered lines. All other lines tested were as resistant to the pathogens as normal sorghums. These results showed that bmr29 and bmr31 can be used to develop varieties and hybrids resistant to charcoal rot under drought while improving sorghum for forage and bioenergy uses through reduced lignin. Increased stalk rot resistance combine with reduced lignin will benefit forage sorghum producers through increased harvestable yield with improved forage quality.
3. Identifying changes to the human gut microbiome in response to waxy and normal sorghum grain in mouse and in vitro models. Grain-containing mutations in the Granule Bound Starch Synthase gene also referred to as ‘waxy’, have altered starch composition. This altered starch composition affects the physical properties of the starch and increases its digestibility. ARS and University of Nebraska researchers in Lincoln, Nebraska, examined the effects of normal and waxy sorghum grain on the human colon microbiome using in vitro experiments and mouse models. The normal sorghum starch was more resistant to digestion, which led to increased levels of bacteria beneficial to human health compared to waxy grain. Adding starch from normal sorghum to the waxy grain restored the growth of these beneficial bacteria in the experiments. The conclusion was waxy starch has potentially undesirable effects on the human gut microbiome. Examining the effects of plant traits on the human microbiome opens new avenues to improve sorghum for human health.
Review Publications
Funnell-Harris, D.L., Sattler, S.E., Toy, J.J., Oneill, P.M., Bernhardson, L.F. 2023. Response of sorghum lines carrying recently identified brown midrib (bmr) mutations to stalk rot pathogens and water deficit. Plant Pathology. https://doi.org/10.1111/ppa.13702.
Grover, S., Shinde, S., Puri, H., Palmer, N.A., Sarath, G., Sattler, S.E., Louis, J. 2022. Dynamic regulation of phenylpropanoid pathway metabolites in modulating sorghum defense against fall armyworm. Frontiers in Plant Science. 13:1019266. https://doi.org/10.3389/fpls.2022.1019266.
Grover, S., Puri, H., Xin, Z., Sattler, S.E., Louis, J. 2022. Genetic analysis of seed traits in Sorghum bicolor that affect the human gut microbiome. Molecular Plant-Microbe Interactions. 35(9):755-767. https://doi.org/10.1094/MPMI-01-22-0005-R.
Yang, Q., Van Haute, M., Korth, N., Sattler, S.E., Toy, J.J., Schnable, J.C., Benson, A.K. 2022. Genetic analysis of seed traits in Sorghum bicolor that affect the human gut microbiome. Nature Communications. 13:5641. https://doi.org/10.1038/s41467-022-33419-1.
Cardona, J.B., Grover, S., Busta, L., Sattler, S.E., Louis, J. 2022. Sorghum cuticular waxes influence host plant selection by aphids. Planta. 257:22. https://doi.org/10.1007/s00425-022-04046-3.
Zhang, B., Vermerris, W., Sattler, S.E., Kang, C. 2023. A sorghum ascorbate peroxidase with four binding sites has activity against ascorbate and phenylpropanoids. Nature Communications. 192(1):102-118. https://doi.org/10.1093/plphys/kiac604.
Puri, H., Grover, S., Pingault, L., Sattler, S.E., Louis, J. 2023. Temporal transcriptomic profiling elucidates sorghum defense mechanisms against sugarcane aphids. BMC Genomics. 24:441. https://doi.org/10.1186/s12864-023-09529-5.
Cardona, J.B., Grover, S., Bowman, M.J., Busta, L., Sarath, G., Sattler, S.E., Louis, J. 2023. Sugars and cuticular waxes impact sugarcane aphid (melanaphis sacchari) colonization on two different developmental stages of sorghum. Plant Science. 330.Article 111646. https://doi.org/10.1016/j.plantsci.2023.111646.
Zhang, B., Lewis, J., Kovacs, F., Sattler, S.E., Sarath, G., Kang, C. 2023. Activity of cytosolic ascorbate peroxidase (APX) from panicum virgatum against ascorbate and phenylpropanoids. International Journal of Molecular Sciences. 24.Article 1778. https://doi.org/10.3390/ijms24021778.
Yang, Q., Vah Haute, M., Korth, N., Sattler, S.E., Rose, D., Price, J., Beede, K., Ramer-Tait, A.E., Toy, J.J. 2023. The waxy mutation in sorghum and other cereal grains reshapes the gut microbiome by reducing levels of multiple beneficial species. Gut Microbes. 15(1):2178799. https://doi.org/10.1080/19490976.2023.2178799.
